Project 3: Development of Novel Reagents to Identify Xenograft Reactive B Cells Project Summary A shortage of available donor organs is the most critical challenge facing organ transplantation today. Pigs are considered a promising source of replacement tissues. Unfortunately, xenotransplantation, the sharing of organs across species, is not clinically applied because humans exert a strong humoral immune response toward pig tissues. Our long-term goal has been to make pig tissues suitable for use in humans by preventing the binding of human antibodies. The central hypothesis of this project is that pig Major Histocompatibility Complex (MHC) proteins, known as Swine Leukocyte Antigens (SLA) in the pig, contribute to xenoantigenicity. We have shown that patients having antibodies against Human Leukocyte Antigens (HLA) often cross-react with the homologous SLA. We have also found that approximately 25% of the population who lack HLA antibodies also have IgG and IgM capable of binding SLA. Inactivating SLA to eliminate their contribution to human anti-pig immunity is problematic because the SLA are key to helping protect the organ from infection and cancer. The objectives of this grant are to: (i) improve our understanding of the frequency with which patients have anti-SLA antibodies; (ii) determine the origin of B cells which produce those antibodies, and (iii) define their susceptibility to a B-cell depleting therapy (Rituximab). Our approach is innovative because it will develop novel tools that build on similar approaches that have been successful in the setting of allotransplantation. Our rationale is that this knowledge will bring us bring us closer to the use of pigs as an organ source by helping to better match donors with recipients. In Specific Aim 1 we will create recombinant SLA, produce a bead array of these proteins, and use the array to screen 500 human sera for the presence of SLA antibodies. The focus of Specific Aim 2.1 is to screen 25 patients before and after Rituximab treatment with the SLA bead array to determine if those xenoreactive antibodies diminish. Specific Aim 2.2 will identify and characterize SLA-specific B cells to determine if natural antibodies account for some of the SLA reactivity. This technology development will contribute significantly to the ability to match pig donors with human recipients by avoiding or eliminating SLA-specific antibodies. This project will rely on several components of this award to achieve its goals. Core C will provide bead arrays for serum screening and SLA tetramers with which to analyze B cells provided by Core B. Project 2 investigators and Core D will help with the sorting and analyses of tetramer-stained B cells. Project 1 will provide Rituximab samples and data and assist in comparisons to their work to determine if B cell depletion affects SLA-specific antibodies and naturally occurring anti-glycan antibodies similarly.